David Piper

Development of the Predictor hERG Fluorescence Polarization Assay Using a Membrane Protein Enrichment Approach

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

Role of Phosphoinositide 3-OH Kinase p110β in Skeletal Myogenesis

ABSTRACT Phosphoinositide 3-OH kinase (PI3K) regulates a number of developmental and physiologic processes in skeletal muscle; however, the contributions of individual PI3K p110 catalytic subunits to these processes are not well-defined. To address this question, we investigated the role of the 110-kDa PI3K catalytic subunit β (p110β) in myogenesis and metabolism. In C2C12 cells, pharmacological inhibition of p110β delayed differentiation. We next generated mice with conditional deletion of p110β in skeletal muscle (p110β muscle knockout [p110β-mKO] mice). While young p110β-mKO mice possessed a lower quadriceps mass and exhibited less strength than control littermates, no differences in muscle mass or strength were observed between genotypes in old mice. However, old p110β-mKO mice were less glucose tolerant than old control mice. Overexpression of p110β accelerated differentiation in C2C12 cells and primary human myoblasts through an Akt-dependent mechanism, while expression of kinase-inactive p110β had the opposite effect. p110β overexpression was unable to promote myoblast differentiation under conditions of p110α inhibition, but expression of p110α was able to promote differentiation under conditions of p110β inhibition. These findings reveal a role for p110β during myogenesis and demonstrate that long-term reduction of skeletal muscle p110β impairs whole-body glucose tolerance without affecting skeletal muscle size or strength in old mice.

A functional screening of the kinome identifies the Polo-like kinase 4 as a potential therapeutic target for malignant rhabdoid tumors, and possibly, other embryonal tumors of the brain

Malignant rhabdoid tumors (MRTs) are deadly embryonal tumors of the infancy. With poor survival and modest response to available therapies, more effective and less toxic treatments are needed. We hypothesized that a systematic screening of the kinome will reveal kinases that drive rhabdoid tumors and can be targeted by specific inhibitors.

Inhibition of polo-like kinase 4 (PLK4): a new therapeutic option for rhabdoid tumors and pediatric medulloblastoma

Rhabdoid tumors (RT) are highly aggressive and vastly unresponsive embryonal tumors. They are the most common malignant CNS tumors in infants below 6 months of age. Medulloblastomas (MB) are embryonal tumors that arise in the cerebellum and are the most frequent pediatric malignant brain tumors. Despite the advances in recent years, especially for the most favorable molecular subtypes of MB, the prognosis of patients with embryonal tumors remains modest with treatment related toxicity dreadfully high. Therefore, new targeted therapies are needed. The polo-like kinase 4 (PLK4) is a critical regulator of centriole duplication and consequently, mitotic progression. We previously established that PLK4 is overexpressed in RT and MB. We also demonstrated that inhibiting PLK4 with a small molecule inhibitor resulted in impairment of proliferation, survival, migration and invasion of RT cells. Here, we showed in MB the same effects that we previously described for RT. We also demonstrated that PLK4 inhibition induced apoptosis, senescence and polyploidy in RT and MB cells, thereby increasing the susceptibility of cancer cells to DNA-damaging agents. In order to test the hypothesis that PLK4 is a CNS druggable target, we demonstrated efficacy with oral administration to an orthotropic xenograft model. Based on these results, we postulate that targeting PLK4 with small-molecule inhibitors could be a novel strategy for the treatment of RT and MB and that PLK4 inhibitors (PLK4i) might be promising agents to be used solo or in combination with cytotoxic agents.

Abstract LB-116: Functional genomics screening using LentiArray™ CRISPR libraries and CellSensor™ assays

Identifying and validating targets that underlie disease mechanisms and can be addressed to provide efficacious therapies remains a significant challenge in the drug discovery and development process. Use of siRNA and shRNA to knock-down RNA and suppress gene function, have provided insights into mechanism of action, but depending on the nature of the targets, cells, biology and end-point assays these approaches may suffer variously from their transient nature, design complexity, incomplete knock-down or off-target effects. The use of CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease and guide RNA (gRNA) provides a strong alternative that can produce long-lasting impact, straightforward design, knock-out of genes and increased specificity. A number of laboratories have already published reports demonstrating how pools of gRNA can be delivered to cells and “hits” can be established through enrichment or depletion of cells following a “survival” assay and identified by sequencing the introduced gRNAs in the remaining cell population. Here we demonstrate a knock-out screening approach that utilizes the Invitrogen™ LentiArray™ CRISPR library to interrogate the impact of individual gene knock-outs on the NFκB pathway as measured by a functional cell-based assay. We describe the library design concepts, the assay development, initial screening results and validation of specific identified hits. The gRNAs are designed to primarily 5’ coding exons of a target gene using our CRISPR design tool to maximize knock-out efficiency and minimize off-target effects. Each gRNA is delivered as a separate lentiviral particle including an antibiotic-resistant marker and each gene is targeted by 4 gRNAs per well, delivered in a 96-well plate. We tested the approach using a library that targets the human kinome and developed a loss-of-function assay using our CellSensor® NF-κB-bla ME180 cell line, which is based on the ratiometric blue/green reporter assay and easily enables identification of genomic targets associated with the NF-κB pathway. We elucidate the key factors in developing a robust assay including both transduction and assay optimization to achieve the highest levels of transduction efficiency and assay window. Using these optimized parameters, we screened the Invitrogen™ LentiArray™ CRISPR kinome library that targets >800 kinases and demonstrate how we followed-up on and validated a subset of the identified hits. We expect these approaches to be scalable to the entire human genome and portable to multiple cell types and end-point assays including both high-throughput plate-based assays and high-content imaging based assays. Citation Format: Chetana M. Revankar, Justin Wetter, Julia Braun, Natasha Roark, Veronica Magnon, LaiYee Wong, Yanfei Zou, Namritha Ravinder, Jian-Ping Yang, Jonathan Chesnut, David Piper. Functional genomics screening using LentiArray™ CRISPR libraries and CellSensor™ assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-116. doi:10.1158/1538-7445.AM2017-LB-116

Abstract LB-105: High-throughput target identification using CRISPR/Cas9

One of the major challenges of the drug discovery process is the identification of novel, validated targets, whose pharmacological modulation may yield the desired therapeutic outcomes. The use of CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease and guide RNA can potentially impact the entire drug discovery process from generation of models based on clinical findings through to the target identification. The ability of CRISPR/Cas9, to efficiently and precisely edit a cell9s DNA and introduce a complete genetic knockout, while minimizing off-target effects, offers an improved approach to target identification. Moreover, the ability to scale this approach through the generation of genome wide CRISPR libraries, coupled with the use of lentiviral delivery methods enables high-throughput (HTP) loss-of-function screens to be performed rapidly and identify genes whose activity is important for the specific endpoint being measured. For example, in this study, we have used CellSensor® cell lines targeting various signaling pathways (AP-1, c-Fos and NF-kB) to identify key targets critical to these pathways. Transcription factors like AP-1, c-Fos and NF-kB have shown to play an important role in cancer initiation and progression. Using the key signaling molecules along these pathways as controls we have data that supports HTP target identification. Furthermore, we will present our results from screening of a subset of a CRISPR library targeting 160 different kinases. Eventually, we plan to screen the entire CRISPR library targeting 750 kinases with 4 gRNA per gene against each of these signaling pathways. Similar screenings can be performed using other functional assay formats, like the cell health assays or high content imaging. These screenings can provide a wealth of data on the normal functioning of a target and in turn, should yield better validated targets for progression into full drug discovery. Ultimately, the hits from the HTP screenings can be followed up by using CRISPR technology to generate animal knockout models that would support translating the screen to the pre-clinical trials. This in turn could provide better correlation to the clinical setting and thereby reduce candidate drug attrition. Citation Format: Chetana M. Revankar, Julia Braun, LaiYee Wong, Justin Wetter, Jian-Ping Yang, Namritha Ravinder, Jon Chesnut, David Piper. High-throughput target identification using CRISPR/Cas9. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-105.